U.S. patent application number 11/305301 was filed with the patent office on 2007-06-21 for calibrating dispensing device performance for complex and/or non-aqueous liquids.
This patent application is currently assigned to Artel. Invention is credited to Keith J. Albert, John Thomas Bradshaw, Alex L. Rogers.
Application Number | 20070141709 11/305301 |
Document ID | / |
Family ID | 38174133 |
Filed Date | 2007-06-21 |
United States Patent
Application |
20070141709 |
Kind Code |
A1 |
Albert; Keith J. ; et
al. |
June 21, 2007 |
Calibrating dispensing device performance for complex and/or
non-aqueous liquids
Abstract
A method for precisely and accurately determining the volume of
a test liquid aliquot which can be used to calibrate a liquid
delivery device. The method is particularly useful for determining
the volume of an aliquot of a test liquid which is complex or
non-aqueous, or is both complex and non-aqueous. The test-stock
mixture contains at least the test liquid and a stock solution
including a first dye, the first dye having absorbance
characteristics which are measurably distinguishable from the
absorbance characteristics of a second dye. The test-stock mixture
is mixed with a diluent including the second dye to form a sample
solution to be used to measure absorbances of the first dye and the
second dye. The volume of the test-stock mixture aliquot is
calculated by using a formula based on the Beer-Lambert law which
incorporates the measured absorbance values and a dilution
characteristic of the test-stock mixture. The test liquid and the
stock solution are combined gravimetrically in a controlled ratio
so that the flow characteristics of the test-stock mixture
correspond to the flow characteristics of the test liquid and
without affecting the ability to distinguish the absorbance
characteristics between the first dye and the second dye.
Inventors: |
Albert; Keith J.; (Auburn,
ME) ; Bradshaw; John Thomas; (Gorham, ME) ;
Rogers; Alex L.; (Gray, ME) |
Correspondence
Address: |
CHRIS A. CASEIRO
VERRILL DANA, LLP
ONE PORTLAND SQUARE
PORTLAND
ME
04112-0586
US
|
Assignee: |
Artel
Westbrook
ME
|
Family ID: |
38174133 |
Appl. No.: |
11/305301 |
Filed: |
December 16, 2005 |
Current U.S.
Class: |
436/8 ;
702/22 |
Current CPC
Class: |
G01N 35/1016 20130101;
Y10T 436/25625 20150115; G01F 25/0092 20130101; G01N 21/25
20130101; Y10T 436/25 20150115; Y10T 436/10 20150115; G01N 1/38
20130101 |
Class at
Publication: |
436/008 ;
702/022 |
International
Class: |
G01N 31/00 20060101
G01N031/00; G06F 19/00 20060101 G06F019/00 |
Claims
1. A system to create a test-stock mixture to be used in the
calibration of a liquid handling device, the test-stock mixture
including a test liquid, the system comprising: a. a stock solution
including a first dye with a known absorbance characteristic; and
b. instructions for mixing the test liquid with the stock solution
to form the test-stock mixture in a ratio of test liquid to stock
solution sufficient so that the flow characteristics of the
test-stock mixture correspond to the flow characteristics of the
test liquid without affecting the ability to distinguish the
absorbance characteristics between the first dye and a second dye,
the second dye having a known absorbance characteristic different
from the absorbance characteristic of the first dye.
2. The system of claim 1 wherein the instructions include
gravimetrically measuring the mass ratio of the test liquid to the
stock solution.
3. The system of claim 1 wherein the stock solution includes the
second dye.
4. A method to create a test-stock mixture to be used in the
determination of a volume of the test-stock mixture to be dispensed
from a liquid handling device to be calibrated, the test-stock
mixture including a test liquid of interest, the method comprising
the step of mixing the test liquid with a stock solution, which
stock solution includes a first dye with a known absorbance
characteristic, to form the test-stock mixture in a ratio of test
liquid to stock solution sufficient so that the flow
characteristics of the test-stock mixture correspond to the flow
characteristics of the test liquid without affecting the ability to
distinguish the absorbance characteristics between the first dye
and a second dye, the second dye having a known absorbance
characteristic different from the absorbance characteristic of the
first dye.
5. The method of claim 4 wherein the step of mixing includes the
step of mixing the test liquid and the stock solution in a weight
ratio as a function of the density of the stock solution and the
density of the test liquid.
6. The method of claim 4 further comprising the step of calculating
a dilution ratio of the stock solution.
7. The method of claim 6 further comprising the step of determining
a z-correction factor for fluctuations in density values of the
test liquid and the stock solution.
8. The method of claim 4 wherein the stock solution includes the
second dye.
9. The method of claim 4 wherein the known absorbance
characteristic of the first dye of the stock solution includes an
absorbance per pathlength of the first dye, the method further
comprising the step of determining a revised absorbance per
pathlength of the first dye in the test-stock mixture.
10. The method of claim 4 wherein the known absorbance
characteristic of the second dye includes an absorbance per
pathlength of the second dye, the method further comprising the
step of determining a revised absorbance per pathlength of the
second dye in the test-stock mixture.
11. A method for determining the volume of a liquid aliquot to be
dispensed from a handling device, comprising the steps of: a.
making a test-stock mixture by mixing a test liquid having a known
weight and a known density value with a stock solution having a
known weight and a known density value, the stock solution
including a first dye with a known absorbance characteristic,
wherein the known weight of the first stock solution and the known
weight of the test liquid are selected in a ratio so that the flow
characteristics of the test-stock mixture correspond to the flow
characteristics of the test liquid without affecting the ability to
distinguish the absorbance characteristics between the first dye
and a second dye, the second dye having a known absorbance
characteristic different from the absorbance characteristic of the
first dye; b. adding an aliquot of the test-stock mixture to a
liquid holder; c. adding a diluent to the test-stock mixture in the
liquid holder to produce a sample solution, the diluent including
the second dye only; d. measuring the absorbance values of the
first dye and the second dye in the sample solution at a first
wavelength and a second wavelength; and e. determining the volume
of the test-stock mixture aliquot by using the measured absorbance
values of the sample solution and the absorbance per pathlength of
either or both of the first dye and second dye.
12. The method of claim 11 wherein the liquid holder is a
multi-well plate.
13. The method of claim 11 wherein step (e) is performed by using
computer-executable software stored on a computer-readable medium,
the computer-executable software being capable of calculating
volume of the test-stock mixture aliquot based upon absorbance
values measured by using the method.
14. The method of claim 11 wherein the liquid holder is in the
shape of a truncated cone and wherein the step of determining
includes the step of calculating the volume of the aliquot by the
equation V S = .pi. D 2 4 a .lamda. 1 ( A .lamda. 1 ) + .pi. D tan
.times. .times. .theta. 2 a .lamda. 1 a .lamda. 2 ( A .lamda. 1 A
.lamda. 2 ) + .pi. tan .times. .times. .theta. 3 a .lamda. 1 a
.lamda. 2 2 ( A .lamda. 1 A .lamda. 2 2 ) ##EQU8## where (V.sub.S)
is the volume of the aliquot of test-stock mixture, D is the
diameter of the liquid holder, O is the taper angle of the liquid
holder, (A.sub..lamda.1) is the measured absorbance of the first
dye at the first wavelength, (A.sub..lamda.2) is the measured
absorbance of the second dye at the second wavelength,
(.alpha..sub..lamda.1) is the absorbance per unit pathlength of the
first dye at the first wavelength, and (.alpha..sub..lamda.2) is
the absorbance per unit pathlength of the second dye at the second
wavelength.
15. The method of claim 11 wherein the liquid holder is in the
shape of a truncated square-based pyramid and wherein the step of
determining includes the step of calculating the volume of the
aliquot by the equation V S = W B a .lamda. 1 ( A .lamda. 1 ) + W B
( W T - W B ) h a .lamda. 1 a .lamda. 2 .times. ( A .lamda. 1 A
.lamda. 2 ) + ( W T - W B ) 2 3 h 2 a .lamda. 1 a .lamda. 2 2 ( A
.lamda. 1 A .lamda. 2 2 ) ##EQU9## where (V.sub.S) is the volume of
the aliquot of test-stock mixture, (A.sub..lamda.1) is the measured
absorbance of the first dye at the first wavelength,
(A.sub..lamda.2) is the measured absorbance of the second dye at
the second wavelength, (.alpha..sub..lamda.1) is the absorbance per
unit pathlength of the first dye at the first wavelength,
(.alpha..sub..lamda.2) is the absorbance per unit pathlength of the
second dye at the second wavelength, (W.sub.B) is the width of the
bottom of the square well, (W.sub.T) is the width of the top of the
square well, and (h) is the height of the entire well.
16. An apparatus for determining the volume of a liquid solution to
be dispensed from a handling device, the apparatus comprising: a.
an analytical balance; b. a spectrophotometer; c. a liquid holder;
d. a central processing unit; and e. computer-executable software
stored on a computer-readable medium, the computer-executable
software being capable of calculating volume of a test-stock
mixture aliquot by using the density values of a test liquid and a
stock solution and by using absorbance values measured by carrying
out a method, the method comprising the steps of. i. making the
test-stock mixture by mixing a known weight of the test liquid with
a known weight of a stock solution including a first dye having a
known absorbance characteristic which is measurably distinguishable
from the known absorbance characteristic of a second dye, ii.
adding an aliquot of the test-stock mixture to a liquid holder;
iii. adding a diluent to the test-stock mixture in the liquid
holder to produce a sample solution, the diluent including the
second dye; iv. measuring the absorbance values of the first dye
and the second dye in the sample solution at a first wavelength and
a second wavelength; and v. calculating the volume of the
test-stock mixture aliquot using the computer-executable software,
the absorbance values, and the density values of the test liquid
and the stock solution.
17. A kit for determining the volume of a test-stock mixture to be
dispensed from a handling device, the test-stock mixture including
a test liquid, wherein the test liquid is a complex and/or
non-aqueous liquid, the kit comprising: a. instructions for
carrying out a method for determining the volume of a liquid
aliquot, comprising the steps of: i. making a test-stock mixture by
mixing the test liquid having a known weight and a known density
value with a stock solution having a known weight and a known
density value, the stock solution including a first dye with a
known absorbance characteristic different from a known absorbance
characteristic of a second dye, wherein the known weight of the
first stock solution and the known weight of the test liquid are
selected in a ratio so that the flow characteristics of the
test-stock mixture correspond to the flow characteristics of the
test liquid without affecting the ability to distinguish the
absorbance characteristics between the first dye and the second
dye; ii. adding an aliquot of the test-stock mixture to a liquid
holder; iii. adding a diluent to the test-stock mixture in the
liquid holder to produce a sample solution, the diluent including
the second dye; iv. measuring the absorbance values of the first
dye and the second dye in the sample solution at a first wavelength
and a second wavelength; and v. determining the volume of the
test-stock mixture aliquot by using the measured absorbance values
and the density values of the test liquid and the stock solution;
and b. the stock solution.
18. The kit of claim 17 wherein the kit includes a test-stock
starter mixture including the test liquid and the stock solution in
place of the stock solution, the test-stock starter solution
including the test liquid and the stock solution in a known weight
ratio.
19. The kit of claim 18 wherein the test-stock starter mixture is
contained in a second liquid holder, wherein the second liquid
holder is sealed to prevent gas exchange between the inside of the
second liquid holder and the outside of the second liquid
holder.
20. The kit of claim 17 including either or both of the first dye
and the second dye in powder form, the kit further comprising
instructions for making either or both of the stock solution and
the diluent using selectable ones of the first dye and the second
dye.
21. The kit of claim 17 wherein the liquid holder is a well of a
multi-well plate.
22. The kit of claim 17 wherein the kit further includes the first
dye.
23. The kit of claim 17 wherein the kit further includes more than
one dye which may be used as the first dye.
24. The kit of claim 17 wherein the kit further includes the second
dye.
25. The kit of claim 17 wherein the kit further includes more than
one dye which may be used as the second dye.
26. The kit of claim 17 wherein the kit further contains
computer-executable software stored on a computer-readable medium,
the computer-executable software being capable of calculating
volume of a liquid aliquot based upon absorbance values measured by
carrying out the method and based upon the density of the test
liquid.
27. The kit of claim 17 wherein the diluent is replaced with a
test-diluent mixture, the test-diluent mixture including the test
liquid and the diluent.
28. The kit of claim 17 wherein the kit further includes a
test-diluent starter mixture including the test liquid and the
diluent, the test-diluent starter solution including the test
liquid and the diluent in a known weight ratio.
29. A system to create a test-diluent mixture to be used in the
calibration of a liquid handling device, the test-diluent mixture
including a test liquid, the system comprising: a. a diluent
including a first dye with a known absorbance characteristic; and
b. instructions for mixing the test liquid with the diluent to form
the test-diluent mixture in a ratio of test liquid to diluent
without affecting the ability to distinguish the absorbance
characteristics between the first dye and a second dye, the second
dye having a known absorbance characteristic different from the
absorbance characteristic of the first dye.
30. The system of claim 29 further comprising a stock solution
including the first dye, the stock solution for mixing with the
test-diluent mixture to form a sample solution, wherein the sample
solution is subject to measurement of the absorbance values of the
first dye and the second dye.
31. The system of claim 29 further comprising a stock solution
including the first dye and the second dye, the stock solution for
mixing with the test-diluent mixture to form a sample solution,
wherein the sample solution is subject to measurement of the
absorbance values of the first dye and the second dye.
32. The system of claim 29 further comprising a test-stock mixture
including the test liquid and a stock solution, the test-stock
mixture including the first dye, the test-stock mixture for mixing
with the test-diluent mixture to form a sample solution, wherein
the sample solution is subject to measurement of the absorbance
values of the first dye and the second dye.
33. The system of claim 29 further comprising a test-stock mixture
including the test liquid and a stock solution, the test-stock
mixture including the first dye and the second dye, the test-stock
mixture for mixing with the test-diluent mixture to form a sample
solution, wherein the sample solution is subject to measurement of
the absorbance values of the first dye and the second dye.
34. A method for determining the volume of an aliquot of a liquid
dispensed into a liquid holder, wherein the liquid is or forms part
of a sample solution, wherein the sample solution includes a first
dye and a second dye to be measured for absorbance at a first
wavelength and a second wavelength, and wherein the liquid holder
is in the shape of a truncated cone, the method comprising the
steps of: a. measuring the absorbance of the first dye and the
second dye at the first wavelength and the second wavelength; and
b. calculating the volume of the aliquot of liquid in the liquid
holder using the equation V S = .pi. D 2 4 a .lamda. 1 .times. ( A
.lamda. 1 ) + .pi. D tan .times. .times. .theta. 2 a .lamda. 1 a
.lamda. 2 .times. ( A .lamda. 1 A .lamda. 2 ) + .pi. tan .times.
.times. .theta. 3 a .lamda. 1 a .lamda. 2 2 ( A .lamda. 1 A .lamda.
2 2 ) ##EQU10## where (V.sub.S) is the volume of the aliquot of
liquid, D is the diameter of the liquid holder, .theta. is the
taper angle of the liquid holder, (A.sub..lamda.1) is the measured
absorbance of the first dye at the first wavelength,
(A.sub..lamda.2) is the measured absorbance of the second dye at
the second wavelength, (.alpha..sub..lamda.1) is the absorbance per
unit pathlength of the first dye at the first wavelength, and
(.alpha..sub..lamda.2) is the absorbance per unit pathlength of the
second dye at the second wavelength.
35. A method for determining the volume of an aliquot of a liquid
dispensed into a liquid holder, wherein the liquid is or forms part
of a sample solution, wherein the sample solution includes a first
dye and a second dye to be measured for absorbance at a first
wavelength and a second wavelength, and wherein the liquid holder
is in the shape of a truncated square-based pyramid, the method
comprising the steps of: a. measuring the absorbance of the first
dye and the second dye at the first wavelength and the second
wavelength; and b. calculating the volume of the aliquot of liquid
in the liquid holder using the equation V S = V B a .lamda. 1 ( A
.lamda. 1 ) + W B ( W T - W B ) h a .lamda. 1 a .lamda. 2 ( A
.lamda. 1 A .lamda. 2 ) + ( W T - W B ) 2 3 h 2 a .lamda. 1 a
.lamda. 2 2 ( A .lamda. 1 A .lamda. 2 2 ) ##EQU11## where (V.sub.S)
is the volume of the aliquot of liquid, (A.sub..lamda.1) is the
measured absorbance of the first dye at the first wavelength,
(A.sub..lamda.2) is the measured absorbance of the second dye at
the second wavelength, (.alpha..sub..lamda.1) is the absorbance per
unit pathlength of the first dye at the first wavelength,
(.alpha..sub..lamda.2) is the absorbance per unit pathlength of the
second dye at the second wavelength, (W.sub.B) is the width of the
bottom of the square well, (W.sub.T) is the width of the top of the
square well, and (h) is the height of the entire well.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to a system for determining
the volume of a liquid. More particularly, the present invention
relates to volume dispensing systems which dispense varied
liquids.
[0003] 2. Description of the Prior Art
[0004] Multichannel volume dispensing devices, such as automated
liquid handling (ALH) systems, are widely used in drug discovery
assays and other high-throughput screening processes. Multichannel
volume dispensing devices are also referred to herein as liquid
handlers and as dispensing systems. The performance of these
systems is heavily based on the ability to accurately and precisely
deliver volumes of specific reagents. For instance, because
concentrations of species within an assay are volume-dependent,
assay integrity and the subsequent interpretation of assay results
are directly tied to the accuracy of the delivered volume, which is
ultimately tied to ALH performance. This direct connection between
assay results and ALH performance demonstrates an imperative need
to ensure proper performance of ALH equipment, especially for
critically important assays.
[0005] There is, however, much to consider when choosing the best
method for calibrating dispensing devices. For one, volumes that
must be accurately and precisely delivered are typically small, so
the method of calibration selected must be particularly effective
for measuring small volumes. Further, it is important that results
produced by the method be standardized, or more specifically,
traceable to national standards to achieve consistent volumetric
delivery performance among all dispensing devices regardless of
make, model, manufacturer or location of the device. It is also
important that the method be robust, rapid, and easy to use in the
laboratory by in-house personnel so that the volumetric performance
of the device can be verified frequently and with minimal device
downtime. This need to be efficient is especially critical for
multichannel device calibration, since for these devices,
calibration must be performed not once, but for each channel
separately to assure accuracy and precision within the entire
device. For purposes of the present invention description,
calibration of a device includes verification and/or validation of
the device.
[0006] Prior methods of determining liquid volume, including those
designed to satisfy the aforementioned considerations, are
well-documented. Several of these methods, such as gravimetric,
fluorometric and photometric approaches, have been used to
calibrate liquid handling systems with varying degrees of success.
A thorough description of these methods is presented in U.S. Pat.
No. 6,741,365 issued to Curtis ("the Curtis '365 Patent"), which is
incorporated entirely herein. Although widely used, gravimetric
analysis generally does not provide information about individual
channel performance for multichannel dispensing devices and is
difficult to perform at low volumes. By comparison, a fluorescence
approach gives information about each channel and readily extends
to low volumes. However, the well-known variability inherent in
fluorescent dyes caused by quenching and photobleaching makes an
accurate volume determination difficult to achieve. These
limitations also make standardization of fluorescence approaches
difficult to achieve, and have hindered the development of
internationally recognized fluorescence standards. As with
fluorescence, a photometric approach provides information about
each channel, but more importantly the photometric approach allows
for volume measurements which are traceable to national and
international standards.
[0007] Added to the limitations of these prior methods is the
problem that none is particularly well-suited for calibrating
liquid handling devices to precisely and accurately dispense any,
or all, liquid types. Indeed, the ability of a liquid handler to
properly dispense can vary from liquid-to-liquid. While liquid
handling instruments are capable of dispensing a wide array of
reagent types, it is commonly known that performance parameters can
vary significantly between different solvent types (e.g., aqueous,
organic, serum, etc.). A dispensing device which precisely and
accurately delivers an aqueous-based liquid, water, for example,
may not be as precise and as accurate in dispensing an
organic-based liquid, dimethyl sulfoxide (DMSO), for example. Thus,
a methodology used for aqueous-based samples may dispense a
significantly different amount of volume if employed for an
organic-based solution of different characteristics. Liquid handler
software packages incorporating computational algorithms may
provide users with the ability to adjust dispense methodologies to
compensate for solvent-dependent performance differences. However,
unless an accurate measurement of the liquid handler performance is
collected, these parameters could lead to a false-sense of
performance when a reagent from a different solvent-type is
employed. In particular, prior methods for calibrating delivery
devices are not optimally designed for calibrating liquid handlers
to dispense liquids which are non-aqueous and/or complex. As
discussed herein, the term complex is used to refer to any liquid
type (aqueous, organic, etc.) that has one or more components
and/or additives, such as dyes, other liquid types, salts, sugars,
detergents, surfactants, proteins, bio- or cellular-based
materials, chelating agents, inorganic materials, etc.
[0008] Therefore, what is needed is a system and related method to
precisely and accurately determine the volume of aqueous, complex,
non-aqueous, or complex and non-aqueous liquids that are dispensed
from a liquid handling device. The system should include one or
more of a determination method, an apparatus, and a kit combining
an apparatus and instructions for carrying out the method. The
system and related method should be suitable for combining a liquid
or solution of interest for use in a liquid handler with a liquid
or solution including one or more dyes suitable for photometric or
other volume measurement techniques.
SUMMARY OF THE INVENTION
[0009] The present invention is a method and related system to
create a test-stock mixture, which is subsequently dispensed from a
liquid handling device, to be used in a volume determination for
calibration of the liquid handling device or other devices of
interest. This test-stock mixture enables the system to verify the
performance of the liquid handler by quantifying the volume of
liquid dispensed therefrom. For the purpose of the description of
the present invention, a test liquid forming part of the test-stock
mixture is primarily a complex and/or non-aqueous liquid, but may
also be an aqueous liquid. The test-stock mixture is prepared by
mixing the test liquid, such as DMSO, for example, with a stock
solution containing a first dye and, optionally, a second dye. The
first and second dyes have known absorbance characteristics and are
used in a volume determination process useful in the calibration of
liquid handling devices. The system includes instructions for
mixing the test liquid with the stock solution to form the
test-stock mixture in a controlled ratio with a known dilution
ratio. The controlled mixture ratio produces a controlled, and
therefore known, change in the absorbance per pathlength determined
by an accurate determination of the dilution ratio. The flow
characteristics of the resulting test-stock mixture correspond to
the flow characteristics of the test liquid. In addition, the mix
ratio of the test liquid and the stock solution preferably provides
such flow characteristics without substantially affecting the
ability to distinguish the absorbance characteristics between the
first dye and the second dye in a sample solution under test, the
sample solution including the test-stock mixture (with either the
first dye only or the first and second dye) and a diluent including
the second dye.
[0010] The mixing of the test liquid and the stock solution is
performed gravimetrically so that the mass ratio of test liquid to
stock solution is controlled. The ultimate volume ratio of the test
liquid to stock solution is thereby based on the density values of
all liquids employed to prepare the test-stock mixture. In general,
the resultant test-stock mixture does not appreciably change the
absorbance characteristics of the dyes in the stock solution (such
as by a change in peak absorbance or in the absorbance spectrum,
for example). However, if the mixture of the test liquid and the
stock solution does result in a change in the absorbance
characteristic of either or both of the first dye and the second
dye, a correction factor may be calculated and applied to the
equations used to determine volume. Whether particular dye
absorbance characteristics do or do not change, it is only
necessary that the absorbance characteristics of the first dye and
the second dye are distinguishable from one another.
[0011] The present invention provides a method and related
apparatus for precisely and accurately determining the volume of a
liquid aliquot of the test-stock mixture. In one embodiment of the
apparatus of the present invention, the apparatus is an analytical
balance, a liquid holder, a spectrophotometer, and a computing
system which may be used to carry out the method of volume
determination including the test-stock mixture.
[0012] In multiple embodiments of a kit of the present invention,
the kit includes the instructions for carrying out the method and
one or more stock solutions to make test-stock mixtures, liquid
holders, dyes, and computer-executable software used to carry out
the method for mixing the test liquid and the stock solution in
controlled ratios, and a method for determining volume.
[0013] The present invention also includes a method and related
system to create a test-diluent mixture, which may be used to make
up a volume within a liquid holder including a liquid, such as a
test-stock mixture as described above, for example, to be used in a
volume calibration determination for a liquid handling device. The
test-diluent mixture is prepared by mixing the test liquid with a
diluent including the second dye only. The system includes
instructions for mixing the test liquid with the diluent to form
the test-diluent mixture in a controlled ratio with a known
dilution ratio. The controlled mixture ratio produces a controlled,
and therefore known, change in the absorbance per pathlength
determined by an accurate determination of the dilution ratio. The
mix ratio of the test liquid and the diluent preferably does not
substantially affect the ability to distinguish the absorbance
characteristics between the first dye and the second dye in a
sample solution under test, the sample solution including the
liquid of interest to be dispensed from a liquid handling device
(with either the first dye only or the first and second dye) and
the test-diluent mixture.
[0014] These and other features and advantages of the invention
will be apparent upon review of the following detailed description,
appended drawings and accompanying claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 is a simplified representation of an embodiment of
the apparatus including an analytical balance, a liquid holder, a
spectrophotometer, and a computing system.
[0016] FIG. 2 is a flow diagram showing the principal steps to be
taken for carrying out the method of the present invention for
making the test-stock mixture and measuring absorbance values of a
sample solution including the test-stock mixture.
[0017] FIG. 3 is a flow diagram illustrating the steps of making
three different test-stock mixtures of the Example used in
describing the present invention.
[0018] FIG. 4 is a flow diagram illustrating the steps for
combining the test-stock mixtures of FIG. 3 with a diluent to make
sample solutions used to determine volume dispensed from a liquid
holder.
[0019] FIG. 5 is one representative image of information displayed
on a computer display of the apparatus prior to carrying out the
dual-dye, dual wavelength ratiometric photometry method.
[0020] FIG. 6 is one representative image of information displayed
on a computer display of the apparatus during the course of
carrying out the dual-dye, dual wavelength ratiometric photometry
method.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0021] The present invention is an apparatus and method for precise
and accurate determination of the volume of a liquid aliquot of a
test-stock mixture formed by mixing together a test liquid of
interest with a stock solution including a first dye or a first dye
and a second dye. The present invention includes a system and
arrangement for volume determination of a sample solution including
the test-stock mixture dispensed and a diluent including the second
dye. The test liquid used in the volume determination is as
primarily described herein a complex and/or non-aqueous liquid as
previously defined. However, the test liquid may be any liquid of
choice, including an aqueous liquid, that is of interest in the
calibration of a device. The present invention includes an optional
kit to aid in performing some or all steps of the method
described.
[0022] The test liquid may be, but is not limited to being, a
liquid or liquid solution such as water, glycerol,
2-mercaptoethanol, benzene, toluene, xylene, polyethyleneglycol,
piperidine, formamide, DMSO, phenol, chloroform, methanol, ethanol,
dichloromethane, isopropanol, nonidet P-40, proteinase K, bovine
serum albumin, fetal bovine serum, fetal calf serum, specifically,
and alcohols, detergents, nucleic acid-containing solutions,
protein-containing solutions, and glycerol-containing solutions,
such as, but not limited to, Taq polymerase, DNA restriction
exonucleases, endonucleases and ligases, and other complex or
non-aqueous or complex and non-aqueous substances, generally.
[0023] A volume-determining apparatus 10 of the present invention,
which is shown in FIG. 1, includes as primary components an
analytical balance 15, a liquid holder 20, a spectrophotometer 30,
and a computing system 40 capable of carrying out calculations
defined through computer-executable software. The analytical
balance 15 is used to weigh out multiple liquids as required by the
method as discussed below. Combinations of these liquids then are
mixed together to make different liquid mixtures including one or
more dyes having absorbance characteristics. An aliquot 21 of each
mixture is added to its own separate liquid holder 20. FIG. I shows
only one aliquot 21 of one mixture contained in one liquid holder
20; however, it is to be understood that multiple mixture aliquots
may be added to one or more liquid holders. The liquid holder 20 is
placeable into the spectrophotometer 30. Liquid holders suitable
for making absorbance measurements by spectrophotometer include
those described in the Curtis '365 patent. The spectrophotometer 30
is capable of being instructed to initiate absorbance measurements
on the liquid mixture aliquot 21, or more specifically, the liquid
mixture aliquot mixed with a diluent including a dye, in the liquid
holder 20. These instructions may be carried out through one or
more input devices of the spectrophotometer 30 or through the
computing system 40. The computing system 40 includes one or more
input devices, such as a keyboard 41, a mouse 42, or a combination
thereof, which may be used to input information, control the
spectrophotometer 30 and/or to perform calculations of volume
determination based on the absorbance measurements. Input devices
may also include bar code scanners used to read bar codes
associated with liquid holders and/or liquid holders containing one
or more of test liquids, stock solutions, diluents, and sample
solutions of the present invention. The computing system 40,
including a computer processor 43 and memory storage, is configured
to carry out executable-system instructions for volume
determination. Input information and output information may be
viewed on a computer display 44. Optionally, a local or remote
printer 45 may be employed to print out input information and/or
output information.
[0024] For the purpose of describing the present invention, the
following terms will be used. First, a liquid of interest,
including any aqueous, complex and/or non-aqueous liquid, to be
used for the purpose of determining the volume of an aliquot of
liquid dispensed by a liquid handling device, for example, is
referred to herein as a test liquid. In general, the test liquid
contains no dye subject to absorption measurements at the
wavelengths of interest for the purpose of the present invention.
Second, a solution including the first dye or the first dye and the
second dye subject to absorption measurements, is referred to
herein as a stock solution. Third, a mixture of a test liquid and a
stock solution is referred to herein as a test-stock mixture.
Fourth, a liquid including the second dye subject to absorption
measurements and used to completely or partially fill a liquid
holder, such as the well of a microtiter plate, is referred to
herein as a diluent. Fifth, a mixture of test liquid and a diluent
is referred to herein as a test-diluent mixture. Finally, the
mixture of a test-stock mixture and a diluent, the mixture of a
test liquid and a test-diluent mixture, the mixture of a stock
solution and a test-diluent mixture, or the mixture of a test-stock
mixture and a test-diluent mixture, any of which is the only liquid
subject to absorption measurements for the purpose of the present
invention, is referred to herein as a sample solution.
[0025] Some of the steps of the method of the present invention are
similar to ones described in the Curtis '365 patent, but with
additional and/or different steps accounting for sample solutions
including the test-stock mixture. These steps are carried out using
a test-stock mixture containing a first dye which absorbs at a
first wavelength (.lamda..sub.1) and a second dye which absorbs at
a second wavelength (.lamda..sub.2), a diluent containing the
second dye only, and a microtiter plate with known dimensions of
the wells within the plate. The steps include dispensing an aliquot
of the test-stock mixture into the wells of a microtiter plate,
followed by (or preceded by) dispensing diluent into the same wells
within the microtiter plate. The test-stock mixture and the diluent
are mixed until a homogeneous mixture is achieved, and the
absorbance of the two dyes in the resultant sample solution are
measured.
[0026] The volume of the aliquot of test-stock mixture dispensed
into each well is determined in part based on the Beer-Lambert law
shown in equation (1), which relates the measured light absorbance
(A) of a sample solution to the molar absorptivity (.epsilon.) of
the dye in the sample solution, the concentration (C) of the dye in
the sample solution, and the pathlength of light (l) through the
sample solution: A=.epsilon.Cl (1) If the concentration of dye in a
sample solution is known, then the concentration can be included
with the molar absorptivity to form a new constant, called the
absorbance per unit pathlength (.alpha.), and equation (1) can be
re-expressed as equation (2): A=.alpha.l (2) The photometric
measurements collected for each well containing a sample solution
in the microtiter plate are used to calculate the aliquot volume in
three steps: 1) calculation of the sample solution depth or
pathlength (based on the Beer-Lambert law), 2) calculation of the
total volume of sample solution, and 3) calculation of the aliquot
volume.
[0027] Provided the concentration of the second dye is the same in
the sample solution and in the diluent, equation (2) is used to
determine the liquid depth (l) in each well, which is equivalent to
the pathlength of light passed through the sample solution. By
measuring the absorbance of the second dye at the second wavelength
(A.sub..lamda.2) and incorporating the known absorbance per unit
pathlength of the second dye at the second wavelength
(.alpha..sub..lamda.2), the sample solution depth is determined by
equation (3): l = A .lamda. 2 a .lamda. 2 ( 3 ) ##EQU1##
[0028] Once the sample solution depth is known, the geometrical
equation for the volume of the wells in the microtiter plate can be
used to determine the total volume of sample solution in the wells.
One common well-shape for 96-well microtiter plates is that of a
trunctated cone, however truncated square pyramidal shapes, and
others also are commonly used. By way of example, for a truncated
cone shape the calculation of the total volume (V.sub.T) is based
on the sample solution depth (l) as determined from equation (3),
and the taper angle (.theta.) and diameter (D) of the microtiter
plate wells, as shown in equation (4): V T = .pi. .times. .times. l
.times. D 2 4 + .pi. .times. .times. Dl 2 .times. tan .times.
.times. .theta. 2 + .pi. .times. .times. l 3 .times. tan 2 .times.
.theta. 3 ( 4 ) ##EQU2##
[0029] The calculation of the aliquot or sample volume (V.sub.S) is
based on the total sample solution volume (V.sub.T), the measured
absorbance of the second dye at the second wavelength
(A.sub..lamda.2), the measured absorbance of the first dye at the
first wavelength (A.sub..lamda.1), the quantities of the absorbance
per unit pathlength of the second dye at the second wavelength
(.alpha..sub..lamda.2) and the absorbance per unit pathlength of
the first dye at the first wavelength (.alpha..sub..lamda.1), as
given by equation (5): V s = V T .function. ( a .lamda. 2 a .lamda.
1 ) .times. ( A .lamda. 1 A .lamda. 2 ) ( 5 ) ##EQU3##
[0030] This relationship is established through use of the fixed
concentration of second dye in both the diluent and the test-stock
mixture. By controlling this concentration, the required
calculation for the total volume of sample solution becomes
independent from calculation of the volume of the aliquot of the
test-stock mixture within the sample solution. This allows use of
equation (5) to determine the volume of aliquot of test-stock
mixture in the sample solution, which corresponds to the test-stock
mixture aliquot dispensed by the liquid handler, from the total
volume of sample solution in the well.
[0031] Equations (3), (4) and (5) can be unified into one governing
equation as shown in equation (6): V s = .pi. D 2 4 a .lamda. 1 ( A
.lamda. 1 ) + .pi. D tan .times. .times. .theta. 2 a .lamda. 1 a
.lamda. 2 ( A .lamda. 1 .times. A .lamda. 2 ) + .pi. tan .times.
.times. .theta. 3 a .lamda. 1 a .lamda. 2 2 ( A .lamda. 1 .times. A
.lamda. 2 .times. 2 ) ( 6 ) ##EQU4## Equation (6) applies to
truncated cone-shaped wells in a microtiter plate. An analogous
equation can be developed for wells shaped like a truncated
square-based pyramid. For wells of this shape, the total volume of
sample solution can be determined using the volume equation for a
truncated square pyramid, as given in equation (7). Required inputs
for this equation include: the pathlength of light through the
solution (l, determined by equation (3)), the width of the bottom
of the square well (W.sub.B), the width of the top of the square
well (W.sub.T), and the height of the entire well (h, which is also
the distance between W.sub.B and W.sub.T). V TOTAL = ( l W B 2 ) +
( l 2 W B ( W T - W B ) h ) + ( l 3 ( W T - W B ) 2 3 .times. h 2 )
( 7 ) ##EQU5## Equations (3), (7) and (5) can be combined into one
governing equation for determining the sample volume dispensed into
a truncated square pyramid-shaped well, as shown in equation (8): V
S = W B a .lamda. 1 ( A .lamda. 1 ) + W B ( W T - W B ) h a .lamda.
1 a .lamda. 2 ( A .lamda. 1 A .lamda. 2 ) + ( W T - W B ) 2 3 h 2 a
.lamda. 1 a .lamda. 2 2 ( A .lamda. 1 A .lamda. 2 2 ) ( 8 )
##EQU6##
[0032] It is to be noted that the approaches described above may
also be used in those cases where very low volumes are to be
determined. In such cases, it is not necessary to include the
second dye in the stock solution used in the formation of the
test-stock mixture. Derived equations (6) and (8) remain
applicable, with only the diluent containing the second dye because
such a small volume of the test-stock mixture is added to a
relatively large volume of diluent. In that case, the absence of
the second dye in the sample solution will have a negligible effect
on the total volume calculation, which in turn will result in a
negligible effect on the calculated test-mixture aliquot volume.
The present invention contemplates this optional approach for low
volume determinations.
[0033] One method 100 of the present invention for testing ALH
performance with test liquids is described with reference to FIG.
2. In this method, the first dye and the second dye are included in
the stock solution to aid in determining liquid volume. The first
dye has an absorbance value at a first wavelength which is
measurably distinguishable from an absorbance value of the second
dye at the first wavelength, and the second dye has an absorbance
value at a second wavelength which is measurably distinguishable
from an absorbance value of the first dye at the second wavelength.
The absorbance values of the dyes as contained in the stock
solution are preferably provided by a supplier capable of ensuring
a high degree of accuracy of the values provided. A test-stock
mixture is made by mixing a known weight of a stock solution, which
contains both the first dye and the second dye, with a known weight
of a test liquid, step 110. By determining the mass (weight) of the
test liquid and of the stock solution added, and incorporating the
density of each, the volume of a particular liquid weighed and
added to the mixture forming the test-stock mixture can be
determined using equation (9): Volume = mass density ( 9 ) ##EQU7##
Knowing the volume of the test liquid and the stock solution,
accurately weighing both and/or one or the other and then the
test-stock mixture after the addition of the other, on a
gravimetric balance and using equation (9) allows for an accurate
determination of the dilution ratio of the test liquid by the stock
solution to be calculated from equation (10): Dilution
Ratio=(Volume Stock Solution)/(Volume Stock Solution+Volume Test
Liquid) (10) Accurately knowing this dilution ratio allows for an
accurate calculation of the absorbance per pathlength of either dye
in the test-stock mixture, which allows for accurate determination
of the volume of test-stock mixture dispensed by a liquid handler
under calibration. That is, the dilution ratio is used to calculate
a revised absorbance per pathlength of the first dye and the second
dye through the following equation:
.alpha.'.sub..lamda.=.alpha..sub..lamda.Dilution Ratio (11) where
the new absorbance per pathlength (.alpha.'.sub..lamda.) is
determined as the product of the absorbance per pathlength from the
stock solution, and the Dilution Ratio of that stock solution. This
new or revised absorbance per pathlength is substituted into the
calculations of volume for which dye absorbance per pathlength
values are required.
[0034] The method described above enables determination of suitable
dilution ratios for the purpose of being capable of measuring
absorbance values in specified volume ranges. Additional accuracy
of determination may be achieved by accounting for fluctuations, if
any, in relative test liquid and stock solution densities as a
function of atmospheric conditions. This optional approach is
carried out by determining a z-correction factor (z), which
accounts for the buoyancy factor of air, and density changes as a
function of atmospheric temperature, pressure and humidity. Using
these factors, a more accurate volume can be calculated using
equation (12): Volume=mass(z/Relative Density) (12) The volume
information obtained from equation (12) may then be plugged into
equation (10). The Relative Density is determined through a process
such as the process described in the description herein of the
formation of the stock solution 1 and stock solution 2 of the stock
solutions for the Example.
[0035] Once a test-stock mixture has been prepared, an aliquot of
the test-stock mixture is added to a well of a liquid holder, step
120. A diluent including the second dye is then added to the same
well to make up a portion of the volume in the well, step 130.
Alternatively, the diluent may be added to the well before the
test-stock mixture. The test-stock mixture and the diluent are
mixed together to form a sample solution. The absorbance of the
sample solution in the liquid holder is measured at the first
wavelength and at the second wavelength by a plate reader, step
140. Finally, the absorbance values measured in step 140 are used
to calculate the volume of the aliquot of test-stock mixture, step
150 using the equations previously described herein. It is to be
noted that an alternative of the method shown in FIG. 2 includes
the use of a stock solution containing only the first dye and none
of the second dye. That alternative arrangement is suitable when
the volumes to be determined are relatively small. In that case,
the absence of the second dye in the sample solution is negligible
in the calculation.
[0036] The particular ratios of test liquid to stock solution are
preferably chosen such that the new absorbance per pathlength
carries over to measurable absorbance values of the sample solution
being tested. That is, the concentration of either or both of the
first and second dyes in the sample solution is in a measurable
absorbance range. They are chosen to cover a desired test volume
range, therefore, they may or may not be different for any
particular sample solution from one volume range to another. For
the purpose of this description, the test volume range is based
upon the final absorbance values that will be measured by the plate
reader. Examples of test-stock mixtures and particular ratios are
presented herein. It is to be noted that the ratios are functions
of the particular liquids of choice in the volume determination. In
general, the present invention is directed to the use of any
selectable liquid as the test liquid, and a stock solution of known
characteristics, such as an aqueous stock solution, and combining
them together to form the test-stock mixture having measurable
absorbance values. The resultant test-stock mixture preferably
includes flow characteristics corresponding to the flow
characteristics of the test liquid while maintaining the ability to
distinguish the absorbance values associated with the first dye and
the second dye when the test-stock mixture and diluent are combined
to form a sample solution that is measured for absorbance
characteristics.
[0037] An optional aspect of the present invention is the creation
of a test-stock starter mixture. While the description of the
present invention has been directed primarily to the instructions
for forming the test-stock solution, it is contemplated that a
manufacturer, rather than an end user, may produce and provide a
prepared test-stock starter mixture including a test liquid of
interest to an end user, and a stock solution including the first
dye or the first dye and the second dye. The prepared test-stock
starter mixture may then be combined with a diluent as described
above and the resultant sample solution subject to absorbance
measurements. In that way, the end user may omit the steps of
preparing the test-stock mixture, including making the required
weight measurements, relying instead on the commercial manufacturer
to perform those steps instead.
[0038] The first dye referred to herein may be any compound which
selectively absorbs light, and the second dye referred to herein
may be any other compound which selectively absorbs light, with the
only limitation being that the first dye must have an absorbance
value at a first wavelength which is measurably distinguishable
from an absorbance value of the second dye at the first wavelength,
and the second dye must have an absorbance value at a second
wavelength which is measurably distinguishable from an absorbance
value of the first dye at the second wavelength. Exemplary
compounds which may be used as the first dye and the second dye are
listed in the Curtis '365 patent. In addition, stock solution
options suitable in the formation of the test-stock mixture of the
present invention are also described in the Curtis '365 patent.
[0039] The method of the present invention is more specifically
described with reference to an Example; however, it is not to be
construed as being limited thereto. In particular, while the
following Example describes instruction for determining the volumes
of aliquots of test-stock mixtures including dimethyl sulfoxide
(DMSO) specifically as the test liquid, the method is not to be
construed as being limited to being used to determine the volumes
of aliquots of test-stock mixtures including DMSO only. The method
may be used to determine the volume of an aliquot of any test-stock
mixture including any test liquid of choice. The stock solutions,
diluents, and devices, mixing, measurement and determination
methods described in the article entitled "Multichannel
Verification System (MVS.TM.): A Dual-Dye Ratiometric Photometry
System for Performance Verification of Multichannel Liquid Delivery
Devices" written by several of the present inventors for the
assignee of the present invention and published in the journal JALA
in February 2005 were used to perform part of the steps used in
evaluating the test-stock mixtures of the Example. The contents of
that publication are incorporated herein by reference. The Example
information is presented in Table 2 (for a 96-well plate) and in
Table 3 (for a 384-well plate) herein.
EXAMPLE
[0040] Solutions Used
[0041] In this Example, the volumes of a plurality of aliquots of
mixtures of DMSO test liquid in aqueous stock solution were
determined by using the method of the present invention. As a first
step of this method, two different formulations of stock solution
and three different formulations of test-stock mixture were
prepared. For the purposes of this Example, these five liquids are
specifically referred to as being "stock solution 1", "stock
solution 2", "test-stock mixture B", "test-stock mixture C", and
"test-stock mixture D." Stock solutions 1 and 2 were prepared as
described in the following two paragraphs. It is to be noted that
stock solutions described in the Curtis '365 patent may also be
used in the alternative.
[0042] Stock solution 1 was manufactured to have an absorbance of
739.2 at 520 nm in a 1 cm pathlength cuvette, and no significant
absorbance at 730 nm. This solution was prepared by dissolving only
the first dye, a red dye component, (that absorbs at 520 nm) into a
previously prepared preserved diluent containing no dye component.
The preserved diluent was prepared by dissolving 0.30 grams Proclin
(a preservative) into one liter of deionized water. The freshly
mixed preserved diluent was filtered through a 0.2 micron filter
and stored in a tightly capped glass bottle. Stock solution 1 was
prepared by dissolving 1,005 grams of Ponceau S into the preserved
diluent to a total batch size of one liter. The relative density of
the stock solution 1, as compared to deionized water, was measured
with a pynknometer. The representative absorbance per a 1 cm
pathlength of stock solution 1 at 520 nm was determined by making
an accurate dilution of 0.05 mL stock solution 1 to 30 mL preserved
diluent. The dilution of stock solution 1 and preserved diluent was
thoroughly mixed, capped and allowed to thermally equilibrate. The
absorbance in a 1 cm pathlength cuvette was measured for the
dilution (0.05/30) at 520 nm. The absorbance per pathlength was
determined by applying the absorbance measured for the dilution by
the dilution factor indicated above. The batch stock solution 1 was
adjusted to an absorbance of 739.2.+-.0.25% at 520 nm in a 1 cm
pathlength cuvette.
[0043] Stock solution 2 was manufactured to have a target
absorbance of 75 in a 1 cm pathlength cell at a wavelength of 520
nm, and an absorbance of 0.610 in a 1 cm pathlength cell at a
wavelength of 730 nm. Stock solution 2 was prepared by dissolving
the red dye component (that absorbs at 520 nm) into a previously
prepared diluent containing the second dye, referred to herein as
the blue dye component, (absorbs at 730 nm). The diluent was
prepared by dissolving 0.30 grams Proclin, 4.0709 grams potassium
hydrogen phthalate (KHP, a buffer salt), 1.1384 grams copper
chloride (dihydrate), and 3.8720 grams EDTA
(ethylenediaminetetraacetic acid, tetrasodium salt, a chelator for
the ionic copper) into deionized water, to a total batch volume of
one liter. The relative density of the diluent, as compared to
deionized water, was measured with a pynknometer. The pH of the
diluent was adjusted to a value of 6.0.+-.0.05 pH units by the
addition of either hydrochloric acid (if the pH is higher than
6.05), or sodium hydroxide (if the pH is lower than 5.95). The
absorbance at 730 nm was measured in a 1 cm pathlength cell and
adjusted to 0.610.+-.0.1%. The diluent was filtered through a 0.2
micron filter and stored in a tightly capped glass bottle. Stock
solution 2 was prepared by dissolving 1.47 grams of Ponceau S into
the diluent to a total batch size of 1 liter. The relative density
of this stock solution 2, as compared to deionized water, was
measured with a pynknometer. The representative absorbance per a 1
cm pathlength of the solution at 520 nm was determined by making a
dilution to bring the stock solution 2 to a measurable absorbance
range (e.g., 1 mL stock solution into 29 mL blank solution). The
dilution was thoroughly mixed, capped and allowed to thermally
equilibrate. The absorbance in a 1 cm pathlength cuvette was
measured at 520 nm, and the representative absorbance for the stock
solution 2 was determined by multiplying the dilution absorbance by
the ratio of the stock solution to the precursor. The stock
solution 2 was adjusted to an absorbance of 75.+-.1% at 520 nm in a
1 cm pathlength cuvette, and to an absorbance of 0.6103.+-.0.1% at
730 nm in a 1 cm pathlength cuvette.
[0044] With reference to FIG. 3, test-stock mixture D was prepared
by mixing 7.0 g of stock solution 1 with 22.96 g of essentially
pure DMSO as the test liquid to make a 25% (vol/vol) solution of
stock solution 1 in DMSO. The gram weight of stock solution 1 was
chosen based primarily on the consideration that the gram weight of
stock solution 1 be large enough to make enough test-stock mixture
D to carry out this particular application of the method, and the
gram weight of the DMSO required was calculated by using the
formula, y=3.28(x), wherein x is the gram weight of stock solution
1, and y represents the gram weight of DMSO required to make a 25%
(vol/vol) solution of stock solution 1 in DMSO. The number given in
this example (i.e., "3.28" from the equation y=3.28x) corresponds
to a multiplier needed to make a final test-stock mixture with an
absorbance per pathlength that can be used to test volumes over a
certain range. Using this number allows the user to determine how
much stock solution and how much test liquid are needed to make a
desired amount of test-stock mixture that the user wants to make.
This multiplier is approximately the inverse of the Dilution Ratio
previously described herein. The same concept applies for all
test-stock mixtures examples described herein.
[0045] Test-stock mixture C was prepared by mixing 3.0 g of stock
solution 1 in 29.837 g of essentially pure DMSO as the test liquid
to make a 9.9% (vol/vol) solution of stock solution 1 in DMSO. The
gram weight of stock solution 1 was chosen based primarily on the
consideration that the gram weight of the stock solution 1 be large
enough to make enough test-stock mixture C to carry out this
particular application of the method, and the gram weight of the
DMSO required was calculated by using the formula, y=9.9457(x),
wherein x is the gram weight of stock solution 1, and y represents
the gram weight of DMSO required to make a 9.9% (vol/vol) solution
of stock solution 1 in DMSO.
[0046] Test-stock mixture B was prepared by mixing 6.0 g of stock
solution 2 in 25.92 g of essentially pure DMSO as the test liquid
to make a 20.2% (vol/vol) solution of stock solution 2 in DMSO. The
gram weight of stock solution 1 was chosen based primarily on the
consideration that the gram weight of the stock solution 2 be large
enough to make enough test-stock mixture B to carry out this
particular application of the method, and the gram weight of the
DMSO required was calculated by using the formula, y=4.32(x),
wherein x is the gram weight of stock solution 2, and y represents
the gram weight of DMSO required to make a 20.2% (vol/vol) solution
of stock solution 2 in DMSO.
[0047] The ratio values identified above for the three different
mixtures were selected to achieve test-stock mixtures for DMSO as
the test liquid in order to conform with the volume ranges of
commercially-available aqueous-based stock solutions, such as the
stock solutions designated as MVS Sample Solutions Range D, C and B
available from Artel of Westbrook, Maine. These or other values may
be used for other solvents as test liquids, dependent upon the
particular density of the solvent and its affect, if any, on the
absorbance characteristics of the dye(s). It may be necessary to
conduct trial-and-error evaluations of mix ratios for compatibility
of the test liquid with the selected stock solution, the volume
range(s) of interest, and the ability to distinguish between the
first and second dye. All solutions of the Example were allowed to
equilibrate to within a temperature range of about 18 .degree. C.
to about 25 .degree. C. prior to being used.
[0048] Choice of Liquid Holder and Volume and Type of Alternative
Solution Determined
[0049] A portion of the Example aliquots ranged in volume from 0.4
.mu.l to 49.9 [.mu.l. The absorbances of these aliquots, were
measured in a liquid holder that was a 96-well microtiter plate.
The remainder of the Example aliquots ranged in volume from 0.1
.mu.l to 9.9 .mu.l. The absorbances of these remaining aliquots
were measured in a liquid holder that was a 384-well microtiter
plate. The particular test-stock mixture, either test-stock mixture
B, test-stock mixture C, or test-stock mixture D, used for any
particular aliquot was chosen based on the expected volume of the
aliquot according to the parameters shown in the following Table 1:
TABLE-US-00001 TABLE 1 Volume of Test-Stock Mixture Added
Compatible Test-Stock Mixture 0.4 .mu.L-4.3 .mu.L Test-Stock
Mixture D 96-wells {open oversize brace} 1.8 .mu.L-10.9 .mu.L
Test-Stock Mixture C 9.1 .mu.lL-49.9 .mu.L Test-Stock Mixture B 0.1
.mu.lL-1.1 .mu.L Test-Stock Mixture D 384-wells {open oversize
brace} 0.5 .mu.L-2.8 .mu.lL Test-Stock Mixture C 2.3 .mu.lL-9.9
.mu.L Test-Stock Mixture B
[0050] Referring to Table 1 above, in all cases where ranges of
volumes of test-stock mixture overlap (left column), two test-stock
mixtures are listed as being appropriate for use. For any
particular aliquot having an expected volume which fell within any
of these overlapping ranges, only one of the two appropriate
test-stock mixtures, chosen arbitrarily, was used.
[0051] Determining Volume
[0052] With reference to FIG. 4, determination of all volumes was
performed by using the apparatus of the invention, one embodiment
of which is shown in FIG. 1 and alternatives or detailed versions
of which are described in the JALA publication and the Curtis '365
patent referenced herein. Generally, an aliquot of each test-stock
mixture 21 was dispensed into its own liquid holder 20, which was
an individual well of a microtiter plate. To this aliquot, the
working volume was adjusted to 200 .mu.L in each well of the
96-well plate, or to 55 .mu.L in each well of the 384-well plate,
by adding diluent. For example, if 2 .mu.L of test-stock mixture C
was added to a well within the 96-well plate, 198 .mu.L of diluent
was added to create a working volume of 200 .mu.L, and if 8 .mu.L
of test-stock mixture B was added to a well within the 384-well
plate, 47 .mu.L of diluent was added to create a working volume of
55 .mu.L. Once the working volume was achieved in each well, a
plate shaker device was used to properly mix the sample solutions
formed by the combination of the test-stock mixture and the
diluent. Each plate including a sample solution was placed into the
spectrophotometer 30 connected to a computing system 40 capable of
carrying out volume determination measurements through
computer-executable software.
[0053] Prior to making any absorbance measurements, the software
was run on the computing system 40 and displayed on the computing
system display 44, and pre-absorbance measurement entries were
made. For instance, the user must enter the characteristics of the
test-stock mixture into the software before absorbance measurements
are acquired. In order to use the computer, associated software and
components of the measurement system to determine the dispensed
volume of the test-stock mixture, information required for the
volume determinations must be entered, such as density values of
the test liquid and stock solution, stock solution absorbance per
pathlength, weight of stock solution, and weight of test liquid.
Entering the noted information may be achieved through direct
manual entry, or through local or remote information exchange
techniques including, but not limited to, bar codes on one or more
components of the invention made readily through bar code scanners
connectable to the computing system 40.
[0054] FIG. 5 is a representative image as seen on the computer
display 44 after all such entries were made in the computing system
40. Generally, this image is a first window 200 into which an
arbitrarily-chosen test-stock mixture identifier may be entered
into a box within the window 200 labeled "Alternative Solution ID"
210, an arbitrarily-chosen descriptive identifier may be entered
into a box labeled "Alternative Solution Description", and the lot
number and type of the stock solution used may be entered into
boxes labeled "MVS Stock Solution Lot Number" 220 and "Stock
Solution Type" 225, respectively. The first window 200 also has a
box, which is labeled "Solvent Solution Density (g/mL)" 230, for
entering the density of the test liquid. Other information that is
entered into the first window 200 is the gram weight 235 of the
stock solution and the gram weight 245 of the test liquid (which is
referred to in the image as being a "solvent solution", which for
the purposes of the present invention is any complex and/or
non-aqueous liquid). Once the weights are entered, the software
calculates the test liquid's percent composition 240 of the
test-stock mixture and the stock solution's percent composition 250
of the test-stock mixture, on a volume-to-volume basis.
[0055] Information regarding the identification 310, description
315, and number of channels 320, of the particular dispensing
device used was entered into a second window 300, a representative
example of which is reproduced in FIG. 6. Other information that
was entered into this second window 300 included information
pertaining to plate layout 325, plate layout description 330,
number of plates being used 335, plate identification 340, number
of wells within the plate 345, plate manufacturer identification
350, and type of plate 355. Additionally, the second window 300
provides boxes into which may be entered, at the operator's option,
information regarding operator name 360, notes 365, temperature
370, pressure 375, and humidity 380. Entry of the noted information
may be achieved through direct manual entry, or through local or
remote information exchange techniques including, but not limited
to, bar codes on one or more components of the invention made
readily through bar code scanners connectable to the computing
system 40.
[0056] After the aforementioned information was entered as
described above and shown in FIG. 4, and the test-stock mixtures in
the indicated ratios made, the mixtures were combined with the
diluent in one or more wells of the microtiter plate in the ratios
indicated herein, and the combination mixed within the wells using
a plate shaker (or other means of mixing), to produce sample
solutions for absorbance testing. The computing system 40 was used
to direct the software to make absorbance readings of the sample
solutions. That is, sample solutions including aliquots of
test-stock mixtures B, C and D were measured. These measurements
were specifically performed by placing the multi-well plate in a
spectrophotometer 30, directing the spectrophotometer 30, via the
computing system 40, to measure each well at the first wavelength,
which specifically was 520 nm, and then again at the second
wavelength, which specifically was 730 nm. Once such absorbance
values were measured, the software of the computing system 40
calculated total sample solution volume and then determined the
volume for each test-stock mixture aliquot added to a well. Volume
determination data generated for the plurality of test-stock
mixture aliquots included in this Example are shown below in Tables
2 and 3. TABLE-US-00002 TABLE 2 Three different test-stock mixtures
dispensed into 9 columns of 96- well microtiter plate; three repeat
dispenses performed per test-stock mixture; aliquots dispensed with
8-channel handheld pipette; DMSO- based test-stock Mixtures for D,
C, and B; aqueous diluent (with 2.sup.nd dye) added after
test-stock mixture added to create working volume of 200 .mu.L in
each well; Columns 1-3 test-stock mixture D with target aliquot of
2 .mu.L; Columns 4-6, mixture C with target aliquot of 8 .mu.L; and
Columns 7-9, mixture B with targ et aliquot of 20 .mu.L. Test-Stock
Mixture Test-Stock Mixture Test-Stock Mixture D, 2 uL C, 8 uL B, 20
uL 1 2 3 4 5 6 7 8 9 A 1.948 1.981 1.977 8.089 8.098 8.128 20.23
20.43 20.18 B 1.957 2.035 2.005 8.191 8.151 8.127 20.28 20.39 20.39
C 2.002 2.021 2.009 8.135 8.152 8.138 20.37 20.41 20.31 D 1.973
1.987 1.922 8.096 8.055 8.07 20.23 20.3 20.3 E 2.019 2.038 2.012
8.141 8.128 8.099 20.51 20.41 20.36 F 1.984 2.006 1.993 8.058 8.123
8.128 20.32 20.41 20.25 G 2.01 2.023 2.006 8.104 8.084 8.123 20.32
20.41 20.41 H 2.115 2.103 2.054 8.071 8.133 8.123 20.2 20.32
20.09
[0057] The table below shows the calculated output data for mean,
relative inaccuracy %, standard deviation and CV % for the
different test-stock mixtures dispensed in replicates of three into
a 96-well microtiter plate. TABLE-US-00003 TABLE 3 Test-Stock
Mixture, Target Volume Mixture D, Mixture C, Mixture B, 2 uL 8 uL
20 uL Mean Volume (uL) 2.008 8.114 20.33 Relative Inaccuracy %
0.40% 1.43% 1.65% Standard Deviation 0.043 0.033 0.1 CV % 2.14%
0.41% 0.49%
Calibrated syringes were employed to dispense aliquots of
DMSO-based test-stock mixtures D, C, and B. These aliquot volumes,
as shown in the tables below, were dispensed into individual wells
of a 384-well microtiter plate. Each well was back-filled with
enough volume of diluent to fill each well to 55 .mu.L of total
working volume. The target volumes are shown directly after the
test-stock mixture ID, i.e., test-stock mixture D, 1 .mu.L where 1
.mu.L is the target volume for the volume verification.
[0058] The table shows the raw data for 8 aliquot dispenses from a
calibrated syringe. All test-stock mixtures were dispensed 8 times
into 8 individual wells. In column 1 of the plate, test-stock
mixture D was dispensed; into column 2 of the plate, test-stock
mixture C was dispensed and into column 3, test-stock mixture B was
dispensed. TABLE-US-00004 Test-stock Test-stock Test-stock mixture
D, mixture C, mixture B, 1 uL 2 uL 8 uL A 1.05 2.054 8.91 B 1.01
2.037 8.51 C 1.065 2.024 8.31 D 1.051 2.068 8.31 E 1.035 2.041 8.31
F 1.034 2.067 8.26 G 1.054 2.043 8.3 H 1.054 2.032 8.22
[0059] The table below shows the calculated output data for mean,
relative inaccuracy %, standard deviation and CV % for the
different test-stock mixtures dispensed in replicates of 8 into 8
individual wells of a 384-well microtiter plate. TABLE-US-00005
Test-stock Test-stock Test-stock mixture D, mixture C, mixture B, 1
uL 2 uL 8 uL Menu (uL) 1.044125 2.04575 8.39125 Relative Inaccuracy
% 3.38% 2.29% 5.02% Standard Deviation 0.017166724 0.015962008
0.22611233 CV % 1.64% 0.78% 2.69%
[0060] In another embodiment of the present invention, a
test-diluent mixture is created. Using the techniques described
herein regarding the combination of a test liquid with a stock
solution, the diluent may be used as a substitute for the stock
solution. The resultant test-diluent solution including the second
dye contained in the diluent, may be used in the formation of a
sample solution to be tested for absorbance measurements of the
first and second dyes. A test-diluent mixture is made by mixing a
known weight of a diluent, which contains only the second dye, with
a known weight of a test liquid. By determining the mass (weight)
of the test liquid and of the diluent added together, and
incorporating the density of each, the volume of a particular
liquid weighed and added to the mixture forming the test-diluent
mixture can be determined using equation (9). Knowing the volume of
the test liquid and the diluent, accurately weighing both and/or
one or the other and then the test-diluent mixture after the
addition of the other, on a gravimetric balance and using equation
(9), allows for an accurate determination of the dilution ratio of
the diluent by the test liquid using equation (10). Accurately
knowing this dilution ratio allows for an accurate calculation of
the absorbance per pathlength of the second dye in the test-diluent
mixture, which allows for accurate determination of the volume of
the liquid dispensed by a liquid handler under calibration through
the equations presented herein for a sample solution including the
test-diluent mixture. That is, the dilution ratio is used to
calculate a revised absorbance per pathlength of the second dye
through equation (11). This new or revised absorbance per
pathlength is substituted into the calculations of volume for which
dye absorbance per pathlength values are required.
[0061] The method described above enables determination of suitable
dilution ratios for the purpose of being capable of measuring
absorbance values in specified volume ranges. Additional accuracy
of determination may be achieved by accounting for fluctuations, if
any, in relative test liquid and diluent densities as a function of
atmospheric conditions. This optional approach is carried out by
determining a z-correction factor (z), which accounts for the
buoyancy factor of air, and density changes as a function of
atmospheric temperature, pressure and humidity. Using these
factors, a more accurate volume can be calculated using equation
(12). The test-diluent mixture may include any diluent of interest
including, but not limited to, the diluents described herein and in
the Curtis '365 patent. The test-diluent mixture may be combined
with a stock solution including either the first dye alone or the
first dye and the second dye, or with a test-stock mixture
including either the first dye alone or the first dye and the
second dye.
[0062] An optional aspect of the test-diluent mixture of present
invention is the creation of a test-diluent starter mixture. While
the description of the present invention has been directed
primarily to the instructions for forming the test-stock solution,
it is contemplated that a manufacturer, rather than an end user,
may produce and provide a prepared test-diluent starter mixture
including a test liquid of interest to an end user, and a diluent
including the second dye only. The prepared test-diluent starter
mixture may then be combined with a stock solution, a test-stock
mixture, or a test-stock starter mixture and the resultant sample
solution subject to absorbance measurements. In that way, the end
user may omit the steps of preparing the test-diluent mixture,
including making the required weight measurements, relying instead
on the commercial manufacturer to perform those steps instead.
[0063] Another optional aspect of the present invention is the way
in which either or both of the stock solution and the diluent are
made available to a user making a sample solution to be subjected
to absorbance measurements. While the discussion of the present
invention herein has been directed to the existence of a stock
solution or a diluent in liquid form, it is contemplated that the
user may be provided with either or both of the first dye and the
second dye in a form other than solubilized in a liquid. For
example, it may be provided to the user as a solid, such as a
powder. The user may also be provided with a liquid to be combined
with the separate dye material, and the two mixed together to form
either the stock solution or the diluent. In this optional
arrangement, the user is provided with instructions to produce the
stock solution or the diluent of interest. The instructions include
steps for making the stock solution or the diluent by adding a
known weight of the dye, preferably but not limited to powder form,
with a specified volume of a liquid of choice, such as an aqueous
liquid, a complex liquid, and/or a non-aqueous liquid. The two are
combined to produce a stock solution or a diluent having known
absorbance characteristics of the first dye and the second dye. For
example, the methods for making the stock solution 1 and stock
solution 2 of the Example described herein represent example steps
to be used in the production of a stock solution, or relatedly, a
diluent, using either or both of the first dye and the second
dye.
[0064] A kit of the present invention contains instructions for
carrying out one or more embodiments of the methods described
herein, and also contains one or more items which may be used to
carry out the methods. Such items may be, but are not limited to
being, one or more stock solutions, one or more dyes which may be
used as the first dye or the second dye, one or more diluents, one
or more liquid holders, or computer-executable software. The items
may also include the dyes in solubilized or separate form, with
instructions for a user to produce a stock solution, a diluent, or
both, using a solubilizing liquid of choice. More specific
embodiments of the kit are described in the Curtis '365 Patent.
[0065] The computer-executable software includes computer-readable
signals tangibly embodied on the computer-readable medium, where
such signals define instructions for processing data obtained from
the spectrophotometer. Such instructions may be written in any of a
plurality of programming languages, for example, Java, XML Visual
Basic, C, or C++, Fortran, Pascal, Eiffel, Basic, COBOL, and the
like, or any of a variety of combinations thereof. The
computer-readable medium on which such instructions preferably
reside is readable by the central processing unit of the computing
system. Further, the steps of processing the data obtained from the
spectrophotometer may be performed in alternative orders, in
parallel and serially.
[0066] It is to be understood that various modifications may be
made to the apparatus, the method, and/or the kit without departing
from the spirit and scope of the invention. For example, the steps
of the method may be performed in differing order, one or more
steps may be omitted, and one or more steps may be replaced with
alternative forms thereof. Accordingly, other embodiments are
within the scope of the claims appended hereto.
* * * * *